1. Field of the Invention
The field of this invention is polypeptide autoantibody inhibitors and methods of use thereof.
2. Background
Multiple sclerosis (MS) is a chronic relapsing remitting disorder disease of the central nervous system that affects 350,000 Americans and, second to trauma, is the leading cause of disability among young adults. MS is an immune-mediated disorder characterized pathologically by perivenular white matter infiltrates comprised of macrophages and mononuclear cells (inflammation), and destruction of the myelin sheaths that insulate nerve fibers (demyelination).
Experimental allergic encephalomyelitis (EAE) in rodents has been the most widely employed model for testing of therapies for human MS. These traditional disease models for MS generally have promoted the concept that MS is a T-cell-mediated disorder. However, the autoantigens that serve as targets for the immune attack have not been identified and the molecular mechanisms implicated in myelin damage remain uncertain. While it is clear that CNS inflammation in EAE is initiated by autoagressive T-cells that recognize myelin antigens in the context of class II-MHC molecules, many of the models lack the early demyelinating component of the MS lesion. B-cell activation and antibody responses appear necessary for the full development of EAE and earlier studies on immune mediated demyelination using myelinated cultures of CNS tissue have implicated humoral factors as effector mechanisms. Thus, it is not surprising that rodent EAE has not been a robust predictor of efficacy in humans as fundamental differences in the clinical course, pathology, and immunologic response to myelin proteins distinguish rodent EAE from human MS.
Recently a novel MS-like illness in an outbred nonhuman primate, the common marmoset Callithrix jacchus, has been defined. The marmoset EAE has a prominent, MS-like early demyelinating component which requires the presence of myelin-specific autoantibodies, and has afforded an opportunity to understand the interactions between these antibodies and their target antigens on myelin. Characteristics of the model include: a. Mild clinical signs and a relapsing remitting course similar to MS; b. A primary demyelinating pathology with early gliosis indistinguishable from MS lesions (demyelinating plaques); c. Natural bone marrow chimerism permitting successful adoptive transfer of encephalitogenic (e.g. disease-inducing) T-cell clones and lines; d. Diversity of the encephalitogenic repertoire of T-cells reactive against the major myelin protein myelin basic protein (MBP); e. Different disease phenotypes resulting from immunization with different myelin constituents: in contrast to whole myelin, immunization with MBP produces a non-demyelinating form of EAE; f. Demonstration that demyelination is antibody-mediated but also requires an encephalitogenic T-cell response to facilitate autoantibody access to the nervous system; and, g. A key role of myelin oligodendrocyte glycoprotein (MOG) in plaque formation: adoptive transfer of anti-MOG antibody in non-demyelinating MBP-EAE reproduces fully developed MS-like pathology.
The highly immunogenic properties of MOG ( less than 0.05% of total myelin protein) may be related to its extracellular location on the outermost lamellae of the myelin sheath, where it is accessible to pathogenic antibody in the context of blood brain barrier disruption by encephalitogenic T-cells. The C. jacchus model permits precise identification of cellular and humoral immune responses that result in an MS-like lesion in a species with immune and nervous system genes that are 90-95% homologous to humans. The relevance of this model to human MS is emphasized by the recent finding of strong T-cell and antibody responses to MOG in MS patients.
The present invention is directed to autoantibody inhibitors and methods of use thereof. Accordingly, the invention provides methods and compositions for inhibiting pathogenic binding of an autoantibody to an autoantigen and screening for inhibitors of pathogenic binding of an autoantibody to an autoantigen.
In one aspect, the present invention provides a composition comprising a peptide consisting of residues 28-36, 13-21, 62-74, 27-34 or 40-45 of rat, human or marmoset MOG (SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:2, respectively). In a preferred embodiment the MOG polypeptide is directly joined at its N- and C-termini with other than natural human or marmoset MOG flanking residues.
In another aspect, the present invention provides a method of inhibiting pathogenic binding of a MOG specific autoantibody to MOG or an immunodominant epitope thereof.
In yet another aspect, the present invention provides a method of detecting autoantibodies in a tissue sample. In a preferred embodiment a method of identifying autoantibodies against myelin/oligodendrocyte glycoprotein (MOG) within lesions of human MS and C. jacchus EAE, where they appear to be directly responsible for the disintegration of the myelin sheaths, is provided.
In a further aspect, the present invention provides a method of screening small molecules or candidate agents capable of binding to an autoantigen and thereby inhibit binding of an autoantibody. The method comprises contacting a solution comprising an autoantigen and an autoantibody, incubating under conditions sufficient to allow the reaction to reach equilibrium, and comparing the binding of the autoantibody in the absence of the small molecule inhibitor or candidate agent to the binding of the autoantibody in the presence of the small molecule inhibitor or candidate agent. In a preferred embodiment the small molecules specifically bind at least one immunodominant epitope of the autoantigen.
In yet another aspect of the invention there is provided a method of inhibiting pathogenic binding of an autoantibody to an autoantigen comprising administering to a host subject to pathogenic autoantigen-autoantibody binding-mediated pathology an effective amount of a composition comprising a fragment of an antibody specific for the autoantigen sufficient to specifically bind the autoantigen and competitively inhibit the binding of an autoantigen-specific autoantibody to the autoantigen, wherein the fragment does not comprise a functional Fc portion of the autoantigen-specific antibody. In a preferred embodiment, the autoantigen-autoantibody binding is associated with a demyelinating disease of the central or peripheral nervous system. In a particular embodiment, the disease is associated with pathogenic autoantibody binding, such as MS, lupus, arthritis or diabetes. In more particular embodiments, the autoantigen is a MOG autoantigen and the fragment is a F(abxe2x80x2)2 fragment.
In yet another aspect, the invention also provides methods of screening for an agent which modulates the binding of an autoantibody to an autoantigen. Such methods generally involve incubating a mixture comprising the autoantibody or an auto antibody-specific binding fragment thereof, the autoantigen, and a candidate agent under conditions whereby, but for the presence of said agent, the autoantibody or fragment thereof specifically binds the autoantigen at a reference affinity; detecting the binding affinity of autoantibody or fragment thereof to the autoantigen to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that said agent modulates the binding of the autoantibody or fragment thereof to the autoantigen. In particular embodiments, the autoantibody or fragment thereof is a F(abxe2x80x2)2 fragment; the autoantigen comprises a MOG epitope; and/or the autoantigen comprises a MOG epitope consisting of residues 28-36, 13-21, 62-74, 27-34 or 40-45 of rat, human or marmoset MOG (SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:2, respectively).
The following description and examples are offered by way of illustration and not by way of limitation.
The invention provides methods and compositions for inhibiting pathology associated with the binding of an autoantibody to a MOG polypeptide, such as occurs in MS. The general methods comprise the step of administering to a host, subject to a pathogenic MOG polypeptide-autoantibody binding, an effective amount of a composition comprising a MOG polypeptide-specific antibody fragment not having a functional Fc portion and sufficient to specifically bind the MOG polypeptide and competitively inhibit the binding of the autoantibody to the MOG polypeptide, whereby the pathology is inhibited. In a particular embodiment, the fragment is selected from the group consisting of Fv, F(abxe2x80x2)2, F(ab), F(ab)2 or fragments thereof.
The compositions include pharmaceutical compositions comprising a MOG polypeptide-specific antibody fragment sufficient to specifically bind a natural MOG polypeptide and competitively inhibit the binding of an autoantibody to the MOG polypeptide, wherein the fragment does not comprise a functional Fc portion, and a pharmaceutically acceptable carrier. The compositions may also comprise a MOG tolerogenic T-cell epitope which induces tolerance and acts synergistically with the antibody fragment to inhibit pathology.
In another embodiment, the invention provides methods and compositions for detecting the presence of an autoantibody bound to a first autoantigen in a tissue. These methods generally comprise the steps of contacting the tissue with a second, labeled autoantigen under conditions wherein the autoantibody binds the second autoantigen to form first autoantigen-autoantibody-second autoantigen labeled complexes, and specifically detecting the labeled complexes. The first and second autoantigens are generally the same or at least include epitopes of the same autoantigen. Preferred autoantigens include, but are not limited to myelin oligodendrocyte glycoprotein (MOG), myelin associated glycoprotein (MAG), myelin/oligodendrocyte basic protein (MOBP), Oligodendrocyte specific protein (Osp), myelin basic protein (MBP), proteolipid apoprotein (PLP), galactose cerebroside (GalC), glycolipids, sphingolipids, phospholipids, gangliosides and other neuronal antigens.
In yet another embodiment, the invention provides methods and compositions for detecting MOG polypeptide-specific B-cells. Such methods generally comprise the steps of fractionating blood to obtain an unselected population of B-cells comprising rare MOG polypeptide-specific B-cells, contacting the population with labeled MOG polypeptides under conditions whereby the labeled MOG polypeptides binds the rare MOG polypeptide-specific B-cells to form labeled complexes of the labeled MOG polypeptides and the rare MOG polypeptide-specific B-cells, and specifically detecting the complexes.
In yet another embodiment, the invention provides methods and compositions for screening for a candidate agent to inhibit pathology associated with MOG polypeptide-specific antibody binding to a MOG polypeptide. These methods generally comprise the steps of:
incubating a mixture comprising: the antibody or a MOG-specific fragment thereof, the MOG polypeptide, and a candidate agent,
under conditions whereby, but for the presence of said agent, the antibody or fragment thereof specifically binds the MOG polypeptide at a reference affinity;
detecting the binding affinity of antibody or fragment thereof to the MOG polypeptide to determine an agent-biased affinity,
wherein a diminution of the agent-biased affinity with respect to the reference affinity indicates that said agent inhibits the binding of the antibody or fragment thereof to the MOG polypeptide and provides a candidate agent for inhibiting pathology associated with MOG polypeptide-specific antibody binding to a MOG polypeptide.
In yet another embodiment, the invention provides polypeptides comprising MOG-specific B- and T-cell epitopes, including polypeptides comprising a fragment having N and C ends and consisting of residues 28-36, 13-21, 67-73, 27-34 or 40-45 of human, rat or marmoset MOG (SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:2, respectively), wherein the fragment is directly joined at at least one of the N and C-ends with other than natural human or marmoset MOG flanking residues. Such polypeptides are useful, for example in methods of inhibiting MOG polypeptide-autoantibody binding, such as the general method comprising the step of contacting a mixture of a MOG and an antibody with a polypeptide, whereby the MOG-antibody binding is inhibited.
As used herein, the term xe2x80x9cantibodyxe2x80x9d refers to recombined immune proteins such as T-cell antigen receptors and immunoglobulins, as well as chimeric, humanized or other recombinant antibodies. As used herein, the term xe2x80x9cantibody fragmentxe2x80x9d refers to fragments of antibodies such as Fab, Fabxe2x80x2, F(ab)2, F(abxe2x80x2)2 and Fv or any combination thereof. Fv and fragments thereof may be monovalent or divalent. Fv is also known in the art as a minimal antibody fragment. Methods of making antibody fragments, particularly F(abxe2x80x2) are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference). For example, F(ab), Fv, etc. can also be produced by recombinant technology.
As used herein, xe2x80x9cother than natural human or marmoset MOG flanking residuesxe2x80x9d refers to anything other than residues naturally flanking the recited peptides in the native proteins. For example, other than natural flanking residues includes no flanking residues or flanking residues different from what naturally flanks the recited peptide.
MOG was originally identified by the mouse monoclonal antibody 8.18.C5, raised against rat cerebellar glycoproteins. It is a quantitatively minor protein representing only 0.01 to 0.05% of the total myelin proteins and has no known function within the CNS. MOG is a member of the immunoglobulin (Ig) superfamily, with an immunoglobulin-like, extracellular domain comprised of 121 amino acids containing one glycosylation site (Asn in position 31) and two highly hydrophobic regions that could represent transmembrane domains, for a total length of 224 amino acids. MOG is widely expressed on oligodendrocyte cell bodies and processes, especially on the outermost layers of the myelin sheaths, and may be more readily accessible to antibody attack than intra-cytoplasmic MBP, or intra and inter-membranous proteolipid apoprotein (PLP). In all species studied including C. jacchus, the non-glycosylated, recombinant extracellular domain of MOG (rMOG) which is highly conserved, suffices for sensitizing animals for EAE. In one aspect of the invention, we have identified minimal T-cell and B-cell epitopes, including residues 28-36, 13-21, 62-74, 27-34 or 40-45; natural human and rat MOG sequences (SEQ ID NO:3 and SEQ ID NO:1, respectively) are known in the art; natural marmoset MOG (SEQ ID NO:2) is identical to the human except for the following substitutions: 9S, 13Q, 19A, 20A, 42S, 60E, 75D, 84K, 91P, 112Q, 137F, 148Y and 151H.
The immune response in autoimmune diseases may possess both cellular and humoral components. Our data indicate that the following sequence of events leads to myelin destruction in CNS autoimmune demyelination:
1) Myelin vacuolation caused by soluble mediators (cytokines, antibodies, free radicals), and/or cellular cytotoxicity. A pattern of intramyelinic edema similar to this has also been observed previously in the CNS of rats intoxicated with tri-ethyl tin sulfate and, interestingly, these changes were reversible.
2) Transformation of vacuolated myelin into networks of small vesicles separated by 2-3 layers of altered myelin with a reduced periodicity (5-6 nm). This dramatic transformation appears to be associated with the deposition of MOG-specific IgG and to reflect antibody-mediated damage, possibly due to complement activation, or antibody-dependent cytotoxicity mediated by macrophages that are invariably associated with vesicular myelin disruption. Conceivably, the initial vacuolar lesion renders the myelin membranes accessible to an attack by autoantibodies.
3) Macrophage activation leading to receptor-mediated phagocytosis of the vesiculated myelin debris. This mechanism has been demonstrated previously in MS and in EAE with IgG serving as a ligand between the myelin debris and Fc receptors in clathrin-coated pits on the macrophage surface. This stage of lesion pathogenesis, although antibody-mediated, may be independent of antibody specificity.
As just outlined above, for example, in MS the inflammatory component is T-cell mediated while the demyelinating component appears to be B-cell mediated. Thus, effective treatments should address both components.
The present invention provides compositions comprising the immunodominant epitopes of MOG. The abolition of the peripheral T-cell response by a tolerization protocol to the extracellular portion of recombinant MOG (aa 1-125) (rMOG; rMOG is comprised of residues 1-125 of the extracellular amino terminus of MOG extended by MRGS at the NH2 and ASES(H)6 at the COOH termini) provided the basis for the present inventive epitope-derived peptide compositions. Mapping of the critical MOG epitopes (including 26-38 and 64-72) was accomplished by cloning T-cells from rMOG-immunized animals and by analyzing T-cell and antibody responses to short peptides of MOG in rMOG immunized marmosets.
Mapping of the antibody response to MOG in C. jacchus indicates limited heterogeneity of epitope recognition by autoantibodies. We have identified regions of MOG that are targeted by demyelinating antibodies using linear peptides. The native, serum polyclonal antibodies in rMOG-immunized marmosets are directed against 4 discrete epitopes along the amino acid sequence, aa 13-21, 28-34, 40-45, 65-74 or shorter sequences, most of which are conserved sequences across species. These peptides differ from those identified to date as antibody epitopes in rodents (aa 35-55), however they bind to antibodies present within the network of vesiculated myelin in acute lesions of human MS as shown in the Examples below. Because most antibodies generally recognize discontinuous epitopes on proteins, our analysis methodology provides detailed knowledge of the structure of MOG is needed to fully define the antigenic repertoire of demyelinating antibodies in C. jacchus and humans. Combinatorial libraries were then made in order to generate F(abxe2x80x2)2 fragments with high affinity for MOG capable of competing with pathogenic IgG and of inhibiting complement-mediated and antibody dependent cellular cytotoxicity. These F(abxe2x80x2)2 fragments were tested alone and in combination with T-cell tolerogenic peptides for their ability to prevent and treat disease in C. jacchus. 
A recently identified patient with a progressive spinal cord disorder associated with an IgG monoclonal gammopathy reactive to MOG offered a unique example of the pathophysiologic consequences of an anti-MOG antibody response in a natural experiment. The human monoclonal antibody was adoptively transferred into a C. jacchus with non-demyelinating EAE. Following adoptive transfer the marmoset developed demyelination. Transfer of human IgG in this species is well-tolerated and the blocking ability of F(abxe2x80x2)2 fragments is demonstrated in the adoptive transfer system. The antibody fragments retain their ability to recognize antigenic epitopes yet lack the ability to activate complement or bind macrophages, they coat the autoantigen such that the endogenous autoantibodies are unable to bind a pathological level.
In the preparation of the pharmaceutical compositions of this invention, a variety of vehicles and excipients and routes of administration may be used, as will be apparent to the skilled artisan. Representative formulation technology is taught in, inter alia, Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, Pa., 1995; e.g. Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th Ed., 1996, McGraw-Hill.